Cigarette smoke is considered one of the main risk factors in the development of chronic obstructive pulmonary disease (COPD). Extracellular heat shock protein 70 (Hsp70) stimulates inflammatory responses through activation of Toll like receptors 2 and 4 (TLR2 and TLR4), followed by induction of mitogen-activated protein kinases (MAPKs) pathways, including extracellular signal-regulated kinases (ERK) and p38. Contrary to this, intracellular Hsp70 has an anti-inflammatory role inside the cell. The aim of this study was to assess the effect of extracellular Hsp70, cigarette smoke extract (CSE), lipopolysaccharide (LPS), lipoteichoic acid (LTA) and their combinations on the expression of receptors TLR2 and TLR4 as well as intracellular Hsp70, and the activation of MAPKs in 16HBE and NHBE human bronchial epithelial cells. Cytotoxicity was determined by MTS viability assay after 24 h treatment. The activation of MAPKs (ERK and p38) and the expression of Hsp70 were determined by Western blot analysis after 30 min, 2 and 8 hours treatments. The expression of TLR2 was determined by quantitative polymerase chain reaction (qPCR) after 24 h treatment. The results indicate that lower doses of CSE increase the viability of 16HBE cells, but higher concentrations have a significant cytotoxic effect. 15% CSE increased the viability of NHBE cells. Furthermore, ERK was activated in 16HBE cells by almost all treatments after 30 minutes of exposure as well as by lower doses of CSE, LPS and LTA after 8 h of treatment. p38 was activated by LPS and LTA after 30 minutes as well as by CSE after all time periods examined. Hsp70 showed lower expression after being exposed to LPS and LTA, but CSE mostly increased its expression. In NHBE cells, 15% CSE was the only treatment that had any effect on the activation of MAPKs and expression of Hsp70. Neither LPS nor LTA had a significant effect on mRNA expression of receptor TLR2, while rhHsp70 caused its down-regulation. On the other hand, with rhHsp70 and LTA or LPS combined treatments a significant synergistic effect inducing an increase in mRNA expression of TLR2 was detected compared to untreated cells as well as to cells treated with LPS or LTA alone. CSE caused an increase in TLR2 mRNA expression after all treatments, but there was a suppressive antagonistic effect on TLR2 mRNA expression observed after combined treatment with 15% CSE and rhHsp70 when compared to untreated cells as well as cells treated with 15% CSE alone. Our results confirm the cytotoxic effects of higher concentrations of CSE, while lower concentrations of CSE could cause alterations in lung epithelial cells proliferation. In addition, results show that rhHsp70, LPS, LTA and CSE can activate MAPKs, and this was dependent on cell type, concentration, and duration of the treatment. Furthermore, interactions of rhHsp70, LPS, LTA and CSE can modulate mRNA expression of TLR2. NHBE cells are less susceptible to applied treatments compared to 16HBE cells.